Multisite phosphorylation of the NDC80 complex gradually tunes its microtubule-binding affinity

Microtubule (MT) attachment to kinetochores is vitally important for cell division, but how these interactions are controlled by phosphorylation is not well known. We used quantitative approaches in vitro combined with molecular dynamics simulations to examine phosphoregulation of the NDC80 complex, a core kinetochore component. We show that the outputs from multiple phosphorylation events on the unstructured tail of its Hec1 subunit are additively integrated to elicit gradual tuning of NDC80-MT binding both in vitro and in silico. Conformational plasticity of the Hec1 tail enables it to serve as a phosphorylation-controlled rheostat, providing a new paradigm for regulating the affinity of MT binders. We also show that cooperativity of NDC80 interactions is weak and is unaffected by NDC80 phosphorylation. This in vitro finding strongly supports our model that independent molecular binding events to MTs by individual NDC80 complexes, rather than their structured oligomers, regulate the dynamics and stability of kinetochore-MT attachments in dividing cells.     

Identification of two novel components of the human NDC80 kinetochore complex

Proper kinetochore function is essential for the accurate segregation of chromosomes during mitosis. Kinetochores provide the attachment sites for spindle microtubules and are required for the alignment of chromosomes at the metaphase plate (chromosome congression). Components of the conserved NDC80 complex are required for chromosome congression, and their disruption results in mitotic arrest accompanied by multiple spindle aberrations. To better understand the function of the NDC80 complex, we have identified two novel subunits of the human NDC80 complex, termed human SPC25 (hSPC25) and human SPC24 (hSPC24), using an immunoaffinity approach. hSPC25 interacted with HEC1 (human homolog of yeast Ndc80) throughout the cell cycle and localized to kinetochores during mitosis. RNA interference-mediated depletion of hSPC25 in HeLa cells caused aberrant mitosis, followed by cell death, a phenotype similar to that of cells depleted of HEC1. Loss of hSPC25 also caused multiple spindle aberrations, including elongated, multipolar, and fractured spindles. In the absence of hSPC25, MAD1 and HEC1 failed to localize to kinetochores during mitosis, whereas the kinetochore localization of BUB1 and BUBR1 was largely unaffected. Interestingly, the kinetochore localization of MAD1 in cells with a compromised NDC80 function was restored upon microtubule depolymerization. Thus, hSPC25 is an essential kinetochore component that plays a significant role in proper execution of mitotic events.     

Under Tension: Kinetochores and Basic Research

The Genetics Society of America’s Edward Novitski Prize recognizes an extraordinary level of creativity and intellectual ingenuity in the solution of significant problems in genetics research. The 2015 winner, Sue Biggins, has made significant contributions to our understanding of how chromosomes attach to the mitotic spindle, a process essential for cell division and frequently impaired in cancer. Among other achievements, Biggins was the first to demonstrate that the Aurora B protein kinase is a key regulator of kinetochore function and that chromatin composition and centromere identity can be regulated by histone proteolysis. In 2010, Biggins and her colleagues were the first to purify kinetochores and, using this system, have already made several groundbreaking discoveries about the function and structure of these crucial components of the segregation machinery.Open in a separate windowIt is easy to forget that basic research on a simple model organism has led to many fundamental insights about how cells work and what goes wrong in disease, especially with the continual pressure from funding agencies and institutions to perform translational research. It is also easy to make the mistake of thinking that all major discoveries using model organisms have already been made.In his Novitski prize essay last year, Charlie Boone noted that the yeast Saccharomyces cerevisiae is better understood than any other cell. This year, I am honored to receive the same award for work that exploited yeast’s powerful combination of relative simplicity and strong conservation of function. In a collaborative effort with Chip Asbury’s lab, we reconstituted kinetochore–microtubule attachments that withstand tension in vitro for the first time (Akiyoshi et al. 2010). Our work is an example of how yeast can provide unexpected insights into conserved processes, and why it is important to support scientists in exploring new directions.Many key discoveries about cell division were initially made using budding yeast. Centromeres were first identified and cloned from yeast, and this information was critical to constructing the first artificial chromosome (Clarke and Carbon 1980; Murray and Szostak 1983; Bloom 2015). Over the years, yeast genetic screens have identified most kinetochore components as well as the key pathways that regulate chromosome segregation (for reviews, see Biggins 2013; Malvezzi and Westermann 2014). Cell-cycle checkpoints were first demonstrated in this organism (Weinert and Hartwell 1988), and the majority of conserved spindle checkpoint genes were identified in two seminal genetic yeast screens (Hoyt et al. 1991; Li and Murray 1991).

Isolating intact kinetochores was an intellectual and technical tour-de-force that laid the groundwork for mechanistic and proteomic analysis of kinetochore proteins. Sue Biggins’ perseverance and intellectual creativity in pursuing this question produced extraordinary insights into how kinetochores interact with microtubules.— Needhi Bhalla, University of California, Santa Cruz

It has been known for decades that chromosome segregation in all organisms relies on the tension-dependent stabilization of proper kinetochore–microtubule attachments (for review, see Nicklas and Ward 1994). This behavior was attributed to a protein kinase-mediated error correction mechanism that destabilizes incorrect attachments because they lack tension (for review, see van der Horst and Lens 2014). To ultimately understand how tension regulates the kinase, I decided that we needed a system for directly manipulating tension on kinetochore–microtubule attachments in vitro. However, kinetochores had never been isolated from any organism; we were far from testing the regulation of error correction in vitro.I was trained as a geneticist, not a biochemist. However, the supportive culture at the “Hutch” (Fred Hutchinson Cancer Research Center) helped our lab to take a risk on something new. Bungo Akiyoshi (now at the University of Oxford) developed the first technique with which to purify the core yeast kinetochore (Akiyoshi et al. 2010). We got a lot of advice and support from colleagues at the Hutch with biochemistry expertise, especially from Toshi Tsukiyama. Once Bungo had optimized a protocol by which to purify kinetochores, our next step was to develop a technique for binding these kinetochores to microtubules and putting them under tension. Fortunately, we are located near Chip Asbury’s lab at the University of Washington, which pioneered laser-trapping techniques to study kinetochore proteins (Asbury et al. 2006; Franck et al. 2007; Powers et al. 2009). Together, our labs used the purified kinetochores to reconstitute kinetochore–microtubule attachments under tension. Our reconstitution system does not include the error correction kinase or any additional cellular factors, so we were surprised to find that the kinetochore–microtubule attachments were stabilized directly by tension (Akiyoshi et al. 2010). Discoveries come from unexpected places—this was preliminary work intended as the foundation for analyzing how tension regulates the error-correction kinase.This finding helps to explain one aspect of the long-standing observation that attachments under tension in vivo are stable. We do not yet know whether and how often the aneuploidy that is a hallmark of so many cancers is due to alterations in kinetochore function, but this reconstitution system can now be applied to understanding the properties of kinetochores in other organisms and in cancer cells. We have also started to use our purification technique to address other aspects of kinetochore function and structure (Gonen et al. 2012; London et al. 2012; Sarangapani et al. 2013; London and Biggins 2014; Sarangapani et al. 2014).

It is […] easy to make the mistake of thinking that all major discoveries using model organisms have already been made. —S.B.

When I first started this work, my grant renewal application did not get a fundable score because of the risky nature of the project and the lack of convincing preliminary data. Luckily, I had colleagues at the Hutch who supported our attempts to do something new despite our lack of expertise. Funding agencies often dismiss applications when the investigator isn’t well versed in the necessary skills, and it is difficult for investigators to obtain money to initiate pilot projects. The current movement of the National Institute of General Medical Sciences and other institutes at the National Institutes of Health to fund investigator-initiated research as well as project-based research is a step in the right direction. We also need to promote collaborations that can help move fields forward, to integrate genetics with other disciplines, and to foster an environment where scientists can try something new. Research in model organisms will continue to provide unpredictable insights into biological processes, especially if we stay open minded to the research we fund and we continue to support investigators who take on new endeavors.     

Kinetochores, microtubules and the metaphase checkpoint

A decade ago, kinetochores were generally regarded as rather uninteresting structures that served only to attach mitotic chromosomes to microtubules. In the past few years, however, a number of experiments have belied this view and demonstrated that kinetochores are actively involved in moving chromosomes along the microtubules of the mitotic spindle. Now it appears that in addition to their function in motility, kinetochores act as dynamic and adaptable centres for regulating cell cycle progression through mitosis.     

The NDC80 complex proteins Nuf2 and Hec1 make distinct contributions to kinetochore-microtubule attachment in mitosis

Successful mitosis requires that kinetochores stably attach to the plus ends of spindle microtubules. Central to generating these attachments is the NDC80 complex, made of the four proteins Spc24, Spc25, Nuf2, and Hec1/Ndc80. Structural studies have revealed that portions of both Hec1 and Nuf2 N termini fold into calponin homology (CH) domains, which are known to mediate microtubule binding in certain proteins. Hec1 also contains a basic, positively charged stretch of amino acids that precedes its CH domain, referred to as the "tail." Here, using a gene silence and rescue approach in HeLa cells, we show that the CH domain of Hec1, the CH domain of Nuf2, and the Hec1 tail each contributes to kinetochore-microtubule attachment in distinct ways. The most severe defects in kinetochore-microtubule attachment were observed in cells rescued with a Hec1 CH domain mutant, followed by those rescued with a Hec1 tail domain mutant. Cells rescued with Nuf2 CH domain mutants, however, generated stable kinetochore-microtubule attachments but failed to generate wild-type interkinetochore tension and failed to enter anaphase in a timely manner. These data suggest that the CH and tail domains of Hec1 generate essential contacts between kinetochores and microtubules in cells, whereas the Nuf2 CH domain does not.     

Syndactyly of the toes

The experience gained through the management of 43 patients with syndactyly of the toes is presented. The incidence appears to be similar to that of syndactyly of the fingers. Type 1 syndactyly, or zygodactyly, always presented itself as a cosmetic problem; its correction is occasionally indicated and the procedure used is discussed. Type 2 syndactyly, or polysyndactyly, represents a functional problem and deserves surgical correction. My negative experience with the more complex procedures described for the correction of polysyndactyly is presented as well as my satisfaction with the simpler procedures. Suggestions for management are offered.     

Kinetochores on chromosomes enclosed within the nuclear envelope

The kinetochore plate which develops after nuclear envelope breakdown in normal cells can be seen to be formed on condensed chromosomes still enclosed in the nuclear envelope in fused multinucleate cells where some nuclei show delayed envelope breakdown caused by nuclear interaction. This suggests that neither nuclear envelope breakdown nor assembly of microtubules is directly related to the formation of the kinetochore plate. Furthermore, it can be clearly observed in these cells that the kinetochores do not have any special association with the nuclear envelope.     

Kinetochores and disease: keeping microtubule dynamics in check!

The essential role of microtubules in cell division has long been known. Yet the mechanism by which microtubule attachment to chromosomes at kinetochores is regulated has only been recently revealed. Here, we review the role of kinetochore-microtubule (kMT) attachment dynamics in the cell cycle as well as emerging evidence linking deregulation of kMT attachments to diseases where chromosome mis-segregation and aneuploidy play a central role. Evidence indicates that the dynamic behavior of kMTs must fall within narrow permissible boundaries, which simultaneously allow a level of stability sufficient to establish and maintain chromosome-microtubule attachments and a degree of instability that permits error correction required for accurate chromosome segregation.     

Kinetochores: if you build it, they will come

Successful mitosis depends critically on the segregation of chromosomes by kinetochore microtubules. A recent paper describes a conserved protein network from Caenorhabditis elegans that is composed of three classes of molecules, each of which contributes uniquely to the building of the kinetochore-microtubule attachment site.     

NDC1: a crucial membrane-integral nucleoporin of metazoan nuclear pore complexes

POM121 and gp210 were, until this point, the only known membrane-integral nucleoporins (Nups) of vertebrates and, thus, the only candidate anchors for nuclear pore complexes (NPCs) within the nuclear membrane. In an accompanying study (Stavru et al.), we provided evidence that NPCs can exist independently of POM121 and gp210, and we predicted that vertebrate NPCs contain additional membrane-integral constituents. We identify such an additional membrane protein in the NPCs of mammals, frogs, insects, and nematodes as the orthologue to yeast Ndc1p/Cut11p. Human NDC1 (hNDC1) likely possesses six transmembrane segments, and it is located at the nuclear pore wall. Depletion of hNDC1 from human HeLa cells interferes with the assembly of phenylalanine-glycine repeat Nups into NPCs. The loss of NDC1 function in Caenorhabditis elegans also causes severe NPC defects and very high larval and embryonic mortality. However, it is not ultimately lethal. Instead, homozygous NDC1-deficient worms can be propagated. This indicates that none of the membrane-integral Nups is universally essential for NPC assembly, and suggests that NPC biogenesis is an extremely fault-tolerant process.     

NDC10: a gene involved in chromosome segregation in Saccharomyces cerevisiae

A mutant, ndc10-1, was isolated by anti-tubulin staining of temperature- sensitive mutant banks of budding yeast. ndc10-1 has a defect chromosome segregation since chromosomes remains at one pole of the anaphase spindle. This produces one polyploid cell and one aploid cell, each containing a spindle pole body (SPD. NDC10 was cloned and sequenced and is identical to CBF2 (Jiang, W., J. Lechnermn and J. Carbon. 1993. J. Cell Biol. 121:513) which is the 110-kD component of a centromere DNA binding complex (Lechner, J., and J. Carbon. 1991. Cell. 61:717-725). NDC10 is an essential gene. Antibodies to Ndc10p labeled the SPB region in nearly all the cells examined including nonmitotic cells. In some cells with short spindles which may be in metaphase, staining was also observed along the spindle. The staining pattern and the phenotype of ndc10-1 are consistent with Cbf2p/Ndc10p being a kinetochore protein, and provide in vivo evidence for its role in the attachment of chromosomes to the spindle.     

Inactivation of the F4/80 glycoprotein in the mouse germ line

Macrophages play a crucial role in the defense against pathogens. Distinct macrophage populations can be defined by the expression of restricted cell surface proteins. Resident tissue macrophages, encompassing Kupffer cells of the liver and red pulp macrophages of the spleen, characteristically express the F4/80 molecule, a cell surface glycoprotein related to the seven transmembrane-spanning family of hormone receptors. In this study, gene targeting was used to simultaneously inactivate the F4/80 molecule in the germ line of the mouse and to produce a mouse line that expresses the Cre recombinase under the direct control of the F4/80 promoter (F4/80-Cre knock-in). F4/80-deficient mice are healthy and fertile. Macrophage populations in tissues can develop in the absence of F4/80 expression. Functional analysis revealed that the generation of T-cell-independent B-cell responses and macrophage antimicrobial defense after infection with Listeria monocytogenes are not impaired in the absence of F4/80. Interestingly, tissues of F4/80-deficient mice could not be labeled with anti-BM8, another macrophage subset-specific marker with hitherto undefined molecular antigenic structure. Recombinant expression of a F4/80 cDNA in heterologous cells confirmed this observation, indicating that the targets recognized by the F4/80 and BM8 monoclonal antibodies are identical.